RFLP Analysis Learner Outcomes

This lab simulation can be applied many different ways depending upon the content and focus of your curriculum. Some possible goals and objectives are listed below.

Each student should:

  1. gain a better understanding of one of the applications of DNA analysis.
  2. become more familiar with the function of restriction enzymes.
  3. better understand the theory behind the gel electrophoresis process.
  4. become proficient at running gel electrophoresis equipment.
  5. know what RFLP's are and how they relate to DNA analysis.

Explanation of the RFLP Technique

  1. Only a small segment of DNA is analyzed out of all the DNA from the cells of an individual. This segment of DNA is used as a probe and is analyzed from the DNA of each person in question. (step 5).
  2. The DNA in each sample is digested with the same restriction enzyme(s). Since every person has DNA with slightly different base sequences, some of the restriction sites will be missing or in different locations. Therefore, each person's DNA restriction enzyme digest will produce unique DNA fragment numbers and sizes.
  3. The samples of fragmented DNA are placed side by side in an agar gel, and are then separated by size using electrophoresis.
  4. The double-stranded DNA fragments are chemically denatured into single-strands ("unzipped"), and blotted onto a nylon sheet which fixes their positions and maintains them as single-stranded DNA.
  5. The nylon sheet is washed with a solution containing many copies of a radioactive DNA probe. The probe is a very short, single-stranded DNA molecule that will stick to ("hybridize" with) its complementary sequence wherever it finds it among the DNA fragments on the nylon. (This technique is called Southern Blotting.) This further increases the selectivity of this procedure.
  6. Finally, an x-ray film is exposed to the nylon sheet. When this film is developed, a dark band will appear at each location where the probe hybridized to complementary DNA. These banding patterns will differ from one person to another, again due to the base pair sequence differences between peoples' DNA.